The purpose of this proposal is to continue our ongoing investigation on the regulatory mechanisms of the mouse kappa opioid receptor (KOR) gene expression which is currently funded by an R01. Following the same direction as proposed in the original project, this renewal application further serves to understand the fundamental problems in the control of opioid receptor expression, by using both tissue culture and transgenic mouse models. It is known that opiates exert extensive pharmacological and physiological effects in animals. Like many other drugs, opioids act through specific receptors on target cells. Their very restricted expression in the adult nervous system and during developmental stages suggests that the specificity and the level of opioid receptor expression must be tightly regulated, and raises an important question of the control for their expression in a homeostatic state. In the previous award period, we have selected the KOR gene as a model to address this question, and our data indicate that KOR gene is under a multi-level regulatory circuit that involves positive and negative transcriptional regulatory mechanisms and several post-transcriptional events such as alternative splicing and translation. The negative transcriptional control is mediated through the action of vitamin A hormone (the retinoic acid), whereas alternative splicing generates KOR mRNA isoforms that have distinct RNA stability, and translation efficiency, and can be differentially transported to neuron cell bodies and fibers. In the same direction as the original project, we will continue to address our principle hypothesis that KOR gene is maintained at a homeostatic state by integrating different levels of regulatory events. At the transcriptional level, KOR is regulated in two phases during stem cell differentiation, with an initial negative effect exerted by an endocrine factor, the retinoic acid, followed by unknown positive mechanisms to reactivate KOR gene in neurons. Ultimately, we will learn: a) the molecular/genetic basis of KOR gene transcriptional regulation, particularly in the context of chromatin, b) KOR gene post-transcriptional control with regard to differential mRNA stability and transport, and c) KOR gene regulatory mechanisms extended from cell cultures to animals (transgenic mice). One important feature of this renewal proposal is that we will test our hypotheses in a more physiologically relevant condition by also studying the regulatory events at the chromatin level. Additionally, the application of transgenic mouse models is essential for understanding the genetic basis of pharmacological problems that are related to the opioid receptor system.